NL2031088B1

NL2031088B1 - Locked Nucleic Acid-modified One-step Nested PCR Primers Set and Kit for African Swine Fever Virus

Info

Publication number: NL2031088B1
Application number: NL2031088A
Authority: NL
Inventors: Gou Hongchao; Cai Rujian; Bian Zhibiao; Li Yan; Yang Dongxia; Shi Keda; Li Chunling; Sun Mingfei; Zhai Shaolun; Jiang Zhiyong; Zhang Kunli; song Shuai; Chu Pinpin
Original assignee: Inst Animal Health Guangdong Academy Agricultural Sciences
Priority date: 2022-02-09
Filing date: 2022-02-27
Publication date: 2022-11-30
Also published as: US20230250497A1; CN114410845A

Abstract

Disclosed is a locked nucleic acid-modified one-step nested PCR primers set and kit for detecting African Swine Fever Virus, which relates to the field of molecular biology. Comprised is an outer primers pair, an inner primers pair and a probe, wherein the upstream primer sequence of the outer primers pair is the sequence shown in SEQ ID NO: 1 and the downstream primer sequence of the outer primers pair is the sequence shown in SEQ ID NO: 2. The upstream primer sequence of the inner primers pair is the sequence shown in SEQ ID NO: 3, and the downstream primer sequence of the inner primers pair is the sequence shown in SEQ ID NO: 4. The sequence of the probe is the sequence shown in SEQ ID NO: 5. Based on the nested PCR principle, the invention designs two pairs of nested primers and simultaneously carries out locked nucleic acid modification on the outer primers to improve the Tm value and stability of the outer primers. Two independent cyclic nested amplifications are carried out, with high sensitivity and specificity, easy operation and less cross contamination.

Description

Locked Nucleic Acid-modified One-step Nested PCR Primers Set and Kit for African Swine Fever Virus

TECHNICAL FIELD The invention relates to the field of molecular biology, in particular to Locked Nucleic Acid- modified One-step Nested primers set and a kit for detecting African Swine Fever Virus.

BACKGROUND African Swine Fever (ASF) is an acute and severe infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and wild boars. The disease is rapid and the mortality rate is as high as 90%-100%, which seriously harms China's pig industry and is listed as a class of infectious diseases by China's Ministry of Agriculture. ASFV belongs to African Swine Fever Virus family of double-stranded DNA virus order, belonging to African Swine Fever Virus genus. ASFV has large individual and complex structure, icosahedral structure and a lipid- containing envelope on its surface, and its genome encodes a large number of proteins, which is easy to mutate and has various natural host genotypes. African Swine Fever Virus has strong resistance to the outside world, and it can survive in the environment for a long time. According to reports, it can survive in blood, faeces and tissues for half a year, in infected raw or undercooked pork products for up to three months, and in frozen meat for several years.

Moreover, African Swine Fever Virus has many genotypes and is easy to mutate. China's African classical swine fever vaccine is in the research and development stage, but it will take some time for it to be successfully developed and applied. At present, the main means of preventing and controlling African swine fever is still monitoring and eliminating the source. Therefore, prevention and control depends on early diagnosis and detection with high sensitivity and specificity, and then the source is eradicated, so laboratory diagnosis is particularly important.

At present, the laboratory diagnosis methods of African Swine Fever Virus mainly include antibody detection: fluorescent immunity method, Elisa antibody detection method, nucleic acid detection: fluorescent quantitative PCR method and ordinary PCR method. Fluorescent quantitative PCR and ordinary PCR are one of the routine and rapid detection methods in the laboratory. The detection limit is about 200-1000 copies/reaction. However, there is a time lag between pig farm sampling and laboratory detection, which leads to degradation of nucleic acid, or poor quality of sample collection, improper extraction of nucleic acid, or different sample types and course of disease, which leads to that the viral load of the detected samples may be lower than the detection limit of traditional fluorescent PCR, resulting in false negative results. Digital PCR can be used to detect the virus load in samples, but it often needs independent reagents, equipment and software, as well as professional personnel, and it is expensive.

SUMMARY The purpose of the present invention is to provide a locked nucleic acid-modified one-step nested PCR primers set and kit for detecting African Swine Fever Virus, in order to solve the above-mentioned problems existing in the prior art. Based on the one-step nested PCR principle, the present invention designs two pairs of nested primers, and simultaneously carries out locked nucleic acid modification on the outer-primers to improve the Tm value and stability of the outer-primers, and carries out two independent cyclic nested amplifications, which has higher sensitivity and specificity, easy operation and less cross pollution.

To achieve the above objective, the present invention provides the following scheme: The invention provide a one-step nested PCR primers set modified by locked nucleic acid for detecting African Swine Fever Virus, which comprises an outer primers pair, an inner primers pair and a probe, wherein that upstream primers sequence of the outer primers pair is the sequence shown in SEQ ID NO: 1, and the downstream primer sequence of the outer primers pair is the sequence shown in SEQ ID NO: 2; The upstream primer sequence of the inner primers pair is the sequence shown in SEQ ID NO: 3, and the downstream primer sequence of the inner primers pair is the sequence shown in SEQ ID NO: 4; The second, ninth, thirteenth, sixteenth and eighteenth bases in the upstream primer sequence of the outer primers pair are all locked nucleic acid, and the first, seventh, tenth, thirteenth and seventeenth bases in the downstream primer sequence of the outer primers pair are all locked nucleic acid; The sequence of the probe is the sequence shown in SEQ ID NO: 5.

The invention also provides the application of the locked nucleic acid-modified one-step nested PCR primers set for detecting African Swine Fever Virus in preparing the African Swine Fever virus detection kit.

The invention also provides a kit for detecting African Swine Fever Virus, which comprises the locked nucleic acid-modified one-step nested PCR primers set for detecting African Swine Fever Virus.

Furthermore, in the PCR reaction system of the kit, the concentration of the probe in the reaction system is 100 nmol/l.

Furthermore, the kit also includes Probe Master Mix as well as DNase and RNase-free water.

Furthermore, in the PCR reaction system of the kit, the annealing temperature of the outer primers pair is 67°C.

Furthermore, in the PCR reaction system of the kit, the annealing temperature of the inner primers pair is 59°C.

Furthermore, in the PCR reaction system of the kit, the concentrations of the upstream primer and the downstream primer of the inner primers pair are both 300 nmol/l.

The invention discloses the following technical effects:

The one-step nested fluorescent PCR method modified by locked nucleic acid of the invention has a detection limit of African Swine Fever Virus 100 times higher than the lowest detection limit of ordinary probe fluorescent quantitative PCR, and has good sensitivity.

The locked nucleic acid-modified one-step nested fluorescence PCR method of the invention has intra-group variation coefficient less than 1% and inter-group variation coefficient less than 3%, which shows that the method has good repeatability.

The locked nucleic acid-modified one-step nested fluorescent PCR method of the invention has higher positive detection rate of nucleic acid in clinical samples than ordinary probe fluorescent quantitative PCR and higher sensitivity.

BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and for ordinary technicians in the field, other drawings can be obtained according to these drawings without creative efforts.

Fig. 1 is a schematic design diagram of nested primers and probe in one-step nested fluorescent PCR method modified by locked nucleic acid; Fig. 2 is gradient annealing temperature gel imaging of inner and outer primers, in which A is gradient annealing temperature gel imaging of outer primers and B is gradient annealing temperature gel imaging of inner primers; Fig. 3 shows the amplification curves of inner primers and probe with different concentrations. A is the fluorescence PCR amplification curves of primers with concentration of 200 - 600 nmol/l, and B is the fluorescence PCR amplification curves of probe with concentrations of 100 - 500 nmol/l; Fig. 4 is the standard curves of locked nucleic acid-modified one-step nested fluorescent PCR of African Swine Fever Virus standard plasmid, in which A is the fluorescence amplification curves of locked nucleic acid-modified one-step nested fluorescent PCR, and the order from 1 to 5is: 3x 10% 3x 103, 3 x 102, 3 x 101, 3 x 10° copies/reaction; NC: negative control; B is amplification curves of ordinary fluorescent PCR , and the order from 1 to 4 is: 3 x 10%, 3 x 103, 3 x 102, 3 x 10%; NC: negative control; C is the standard curves of locked nucleic acid-modified one-step nested fluorescence PCR and common probe fluorescence PCR.

DESCRIPTION OF THE INVENTION Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present invention.

It should be understood that the terms used in this invention are only for describing specific embodiments, and are not used to limit the invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Any stated value or intermediate value within the stated range, as well as any other stated value or each smaller range between intermediate values within the stated range, are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary technicians in the field of this invention. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.

Without departing from the scope or spirit of the present invention, it is obvious to those skilled in the art that many modifications and changes can be made to the specific embodiments of the present invention. Other embodiments obtained from the description of the present invention will be obvious to the skilled person. The description and example of that present invention are exemplary only.

The words "including", "include", "have" and "contain" used in this paper are all open terms, that is, they mean including but not limited to.

Example 1 primers and probe design Referring to the nucleic acid sequence of the whole gene of African Swine Fever Virus B646L (P72) protein (Gene ID: 22220311), the full length of the gene sequence is 1941 bp. After sequence alignment of mutant strains, conservative sequences were selected, and nested primers and probe were designed by Oligo 7 as primers and probe of one-step nested fluorescent PCR modified by locked nucleic acid, as shown in Figure 1. Nucleic acid sequence of nested primers and probe are shown in Table 1:

Table 1 Base sequences of one-step nested fluorescent PCR primers and probe modified by locked nucleic acid EE Nuckic acid sequence (5-3) “Outer primer F1 TG(H)GTATTCC(H)TCCCH)GTGH)GC(HTTC (SEQIDNO: 1) Outer primer R1 C(+)CCCAGT(+)AGA(+)CGC(+)AATA(H)TACGC (SEQ ID NO: 2) inner primer F2 ATAGATGAACATGCGTCTGG (SEQ ID NO: 3) inner primer R2 CAAAATCCTCATCAACACCG (SEQ ID NO: 4) Probe T1 FAM-CTGAAAGCTTATCTCTGCGTGGT-BHQ1 (SEQ ID NO: 5) _ Note: (+) indicates the base of this nucleic acid-modified by locked nucleic acid. 5 Locked nucleic acid-modified one-step nested fluorescent PCR method flow, as shown in Figure 1, in the first step, the outer primers F2 and R2 modified by lock nucleic acid were used to amplify the 164 - 762 sites of the sequence, and the 592 bp product fragment was obtained after 10 cycles; In the second step, based on the product fragment amplified in the first step, inner primers F1 and R1 were used to amplify the sequence at positions 377 - 488, and at the same time, they were combined with probe to release FAM fluorescent groups, which could be used by fluorescence PCR equipment to collect signals. Example 2 Optimization of amplification conditions

1. Annealing temperature of outer primers The outer primer F1 and the outer primer R1 were used for conventional PCR amplification. The annealing temperature was set at 66 - 72°C and the gradient was 1°C. Other conditions were the same as those of conventional PCR. The added DNA template was the standard plasmid of African Swine Fever Virus B646L (P72) protein. After the reaction, PCR amplification products were detected by agarose electrophoresis. The results show that the optimum annealing temperature is 67°C

2. Annealing temperature of inner primers The inner primer F2 and the inner primer R2 were used for conventional PCR amplification. The annealing temperature was set at 58 - 66°C and the gradient was 1°C. Other conditions were the same as those of conventional PCR. The added DNA template was the standard plasmid of African Swine Fever Virus B646L (P72) protein. After the reaction, PCR amplification products were detected by agarose electrophoresis. The results show that the optimum annealing temperature is 59°C.

3. Optimization of inner primers concentration One-step nested fluorescence PCR with locked nucleic acid modification at the best annealing temperature, the reaction conditions are: pre-denaturation at 95°C for 5 min; Denaturing at 95°C for 15 s, annealing at 67°C for 30 s, stretching at 72°C for 40 s, and cycling for 10 times; Denaturation at 95°C for 15 s, annealing at 59°C for 30 s, extension at 72°C for 15 s, and cycling for 45 times. Set the inner primers concentrations of 200 nmol/l, 300 nmol/l, 400 nmol/l, 500 nmol/l and 600 nmol/l respectively, and the reaction system is as follows. 2 uM outer primer F2 0.5 pl, 2 uM outer primer R2 0.5 ul, uM inner primer F1 0.4 ul - 1.2 ul, 10 HM inner primer R1 0.4 ul - 1.2 ul, 10 uM probe T1 0.2 ul, Probe Master Mix 10 ul, African Swine Fever Virus plasmid 5 pl, 10 completed to 20 ul with water that is free from DNAse and RNAse. Results As shown in Figure 3 A, the optimal concentration of inner primer F1 and inner primer R1 was 300 nmol/L.

4. Optimization of probe concentration One-step nested fluorescence PCR with locked nucleic acid modification at the best annealing temperature, the reaction conditions are: pre-denaturation at 95°C for 5 min; Denaturing at 95°C for 15 s, annealing at 67°C for 30 s, stretching at 72°C for 40 s, and cycling for 10 times; Denaturation at 95°C for 15 s, annealing at 59°C for 30 s, extension at 72°C for 15 s, and cycling for 45 times. Set the concentration of probe T1 at 100 nmol/l, 200 nmol/l, 300 nmol/l, 400 nmol/l and 500 nmol/l. The reaction system is as follows: 2 UM outer primer F2 0.5 ul, 2 uM outer primer R2 0.5 ul, 10 uM inner primer F1 0.6 pl, 10 uM inner primer R1 0.6 pl, 10 HM probeT1 0.2 pl - 1.0 pl, Probe Master Mix 10 ul, African classical swing fever standard plasmid 5 ul, completed to 20 ul with water that is free from DNAse and RNAse. Results As shown in Figure 3 B, the optimal concentration of probe T1 was 100 nmol/L. Example 3 Establishment of standard curves The standard plasmid of African Swine Fever Virus P72 was reacted according to 3 x 10%, 3x 103 3 x 102, 3 x 10" and 3 x 10° copies/reaction, diluted by 10 times gradient, and nuclease water was used as negative control. According to the reaction system and conditions optimized in Example 2, one-step nested fluorescence PCR modified by locked nucleic acid was carried out to obtain amplification curves of plasmids with different concentrations, and standard curves were drawn. Common probe fluorescence PCR was used as control.

Test results: If the Cq value of fluorescence quantitative PCR is less than or equal to 31 and there is a specific "S" amplification curves, it is judged as positive; If the Cq value of fluorescence quantitative PCR is greater than 31, or there is no specific "S" amplification curves, it will be judged as negative.

Result: One-step nested fluorescence PCR amplification curves modified by locked nucleic acid is shown in Figure 4 A; The fluorescence amplification curves of ordinary probe fluorescence PCR is shown in Figure 4 B. Locked nucleic acid-modified one-step nested fluorescent PCR and common probe fluorescent PCR are shown in Figure 4 C. The results showed that the lowest detection limit of locked nucleic acid-modified one-step nested fluorescence PCR was 3 x 10° copies/reaction. The lowest detection limit of common probe fluorescence PCR is 3 x 10? copies/reaction; The lowest detection limit of locked nucleic acid- modified one-step nested fluorescent PCR is 100 times higher than that of ordinary probe fluorescent PCR.

Example 4 Repeatability Test of locked nucleic acid Modified One-step Nested Fluorescence PCR The standard plasmid of African Swine Fever Virus P72 was reacted according to 3 x 10%, 3x 10% 3x 102, 3x10" and 3 x 10° copies/reaction, diluted by 10 times gradient, and tested with three independent replicates at each concentration. According to the optimized reaction system and conditions in Example 2, one-step nested fluorescence PCR with locked nucleic acid modification was carried out.

Result: As shown in Table 2, the intra-group coefficient of variation was less than 1%, and the inter-group coefficient of variation was less than 3%. The established method has good repeatability and reliable results.

Table 2 Repeatability test of locked nucleic acid-modified one-step nested fluorescent PCR “weg Whingoup™ Eemesngoups (copies/yL) xts Variable xts Variable coefficient (%) coefficient (%) ~ 1.5x10° 18.5840.12 062 OO 18794046 245

1.5x102 21.51+0.10 0.48 21.67 £0.64 2.95

1.5x10! 24.8410.05 0.21 2497 £0.48 1.90

1.5x10° 27.8310.19 0.67 28.2110.14 0.50

1.5x107% 30.08+0.06 0.21 30.66 £0.77 2.50

Example 5 Detection of locked nucleic acid-modified one-step nested fluorescent PCR clinical samples 96 samples of nucleic acids suspected to be suffering from African swine fever were selected for detection, and the nucleic acids of clinical samples were extracted from the pig ward of the Institute of Animal Health, Guangdong Academy of Agricultural Sciences. The method of the present invention is used for detection, the primers and probe are amplified as in Example 1, the reaction conditions and systems are shown in Example 2, and the positive judgment is shown in Example 3; At the same time, common probe fluorescence PCR was used for detection and parallel comparison.

Results: The results of one-step nested fluorescent PCR modified by locked nucleic acid showed that 55 samples were positive and 41 samples were negative, and the positive detection rate was 57.3%. Common probe fluorescent PCR results showed that 47 samples were positive and 49 samples were negative, and the positive detection rate was 48.9%. It shows that locked nucleic acid-modified one-step nested fluorescent PCR is more sensitive than ordinary probe fluorescent PCR.

Table 3 Clinical detection of common probe method and one-step nested fluorescent PCR modified by locked nucleic acid ~~ Onestepnested locked Detection of ASFV nucleic acid fluorescence Routine probe PCR quantification PCR Positive (sample) ~~ 5 41 Negative (sample) 41 49 Total (samples) 96 96 Detection rate of positive 57.3% 48.9% sample The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the embodiments of the present invention.

Sequence List <110> Institute of Animal Health, Guangdong Academy of Agricultural Sciences <120> Locked Nucleic Acid-modified One-step Nested PCR Primers Set and Kit for African Swine Fever Virus <130> SHX-ASFW Primers NL <150> CN 202210121406.X <151> 2022-02-09 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <400> 1 tggtattcct cccgtggctt c 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <400> 2 ccccagtaga cgcaatatac gc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 atagatgaac atgcgtctgg

<210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 caaaatcctc atcaacaccg 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <400> 5 ctgaaagctt atctctgcgt ggt 23

Claims

CONCLUSIESCONCLUSIONS

1. Een set primers voor enkelstaps geneste PCR, die is gemodificeerd met vergrendeld nucleinezuur, voor het opsporen van het Afrikaanse varkenspestvirus, welke set omvat: een paar buitenste primers, een paar binnenste primers en een probe, waarbij — de stroomopwaartse primersequentie van het paar buitenste primers de sequentie is van SEQ ID NO: 1, en de stroomafwaartse primersequentie van het paar buitenste primers de sequentie is van SEQ ID NO: 2; — de upstream-primersequentie van het paar binnenste primers de sequentie is van SEQ ID NO: 3, en de downstream-primersequentie van het paar binnenste primers de sequentie is van SEQ ID NO: 4; — de tweede, negende, dertiende, zestiende en achttiende basen in de stroomopwaartse primersequentie van het paar buitenste primers alle vergrendelde nucleïnezuren zijn, en de eerste, zevende, tiende, dertiende en zeventiende basen in de stroomafwaartse primersequentie van het paar buitenste primers alle vergrendelde nucleïnezuren zijn; — de sequentie van de probe de sequentie is als weergegeven in SEQ ID NO: 5.1. A set of primers for single-step nested PCR modified with locked nucleic acid for the detection of African swine fever virus, comprising a pair of outer primers, a pair of inner primers and a probe, where — the upstream primer sequence of the pair outer primers is the sequence of SEQ ID NO: 1, and the downstream primer sequence of the pair of outer primers is the sequence of SEQ ID NO: 2; - the upstream primer sequence of the inner primer pair is the sequence of SEQ ID NO: 3, and the downstream primer sequence of the inner primer pair is the sequence of SEQ ID NO: 4; — the second, ninth, thirteenth, sixteenth and eighteenth bases in the upstream primer sequence of the outer primer pair are all locked nucleic acids, and the first, seventh, tenth, thirteenth and seventeenth bases in the downstream primer sequence of the outer primer pair are all locked nucleic acids to be; — the sequence of the probe is as shown in SEQ ID NO: 5.

2. Toepassing van set primers voor enkelstaps geneste PCR, die is gemodificeerd met vergrendeld nucleïnezuur volgens conclusie 1 voor het opsporen van Afrikaanse varkenspestvirus bij de bereiding van een samengestelde set voor het opsporen van Afrikaanse varkenspestvirus.Use of single step nested PCR primer set modified with locked nucleic acid according to claim 1 for the detection of African swine fever virus in the preparation of a composite set for the detection of African swine fever virus.

3. Een samengestelde set voor het opsporen van Afrikaanse varkenspestvirus, die de met vergrendeld nucleïnezuur gemodificeerde set primers voor enkelstaps geneste PCR volgens conclusie 1 omvat voor het opsporen van Afrikaanse varkenspestvirus.A composite kit for detecting African swine fever virus comprising the locked nucleic acid modified set of primers for single step nested PCR according to claim 1 for detecting African swine fever virus.

4. De samengestelde set voor het opsporen van Afrikaanse varkenspestvirus volgens conclusie 3, waarbij in het PCR-reactiesysteem van de set de concentratie van de probe in het reactiesysteem 100 nmol/l bedraagt.The composite kit for detecting African swine fever virus according to claim 3, wherein in the PCR reaction system of the kit, the concentration of the probe in the reaction system is 100 nmol/L.

5. De samengestelde set voor het opsporen van Afrikaanse varkenspestvirus volgens conclusie 3, waarbij de set ook probe - mastermix en water dat vrij is van DNAse en RNAse omvat.The composite kit for detecting African swine fever virus according to claim 3, wherein the kit also comprises probe - master mix and water free of DNAse and RNAse.

6. De samengestelde set voor het opsporen van Afrikaanse varkenspestvirus volgens conclusie 3, waarbij in het PCR-reactiesysteem van de set de temperatuur voor het aaneenhechten van het paar buitenste primers 67°C bedraagt.The composite kit for detecting African swine fever virus according to claim 3, wherein in the PCR reaction system of the kit, the temperature for annealing the pair of outer primers is 67°C.

7. De samengestelde set voor het opsporen van Afrikaanse varkenspestvirus volgens conclusie 3, waarbij in het PCR-reactiesysteem van de set de temperatuur voor het aaneenhechten van het paar binnenste primers 59°C bedraagt.The composite kit for detecting African swine fever virus according to claim 3, wherein in the PCR reaction system of the kit, the temperature for annealing the pair of inner primers is 59°C.

8. De samengestelde set voor het opsporen van Afrikaanse varkenspestvirus volgens conclusie 3, waarbij in het PCR-reactiesysteem van de set de concentraties van de upstream primer en de downstream primer van het paar binnenste primers beide 300 nmol/l bedragen.The composite kit for detecting African swine fever virus according to claim 3, wherein in the PCR reaction system of the kit, the concentrations of the upstream primer and the downstream primer of the pair of inner primers are both 300 nmol/L.